Kaleidagraph software

All solvents, used in the synthesis and purification, were anhydrous and HPLC grade, respectively, and were supplied by Romil (Cambridge, UK). All buffer solutions were made by using water with a HPLC purity grade (Romil); phosphate salts (monobasic and dibasic) for buffers preparation, NHS, THF (tetrahydrofuran), uranyl acetate dihydrate and H₂O₂ (30%, v/v) were provided by Fluka; TFE was supplied from Romil. The DCC (N,N′-Dicyclohexylcarbodiimide) as well as HAuCl₄ solution (30% w/w) were obtained from Sigma Aldrich (Taufkirchen, Germany). The electrodes, the electrochemical cell, the alumina and the diamond powder for cleaning the electrodes are from BASi (West Lafayette, IN, USA).
Iron content of MIMO@LA@AuNPs solution was quantified by ICP-MS analysis, using Aurora Bruker M90 instrumentation (Bremen, Germany). Aqua regia digestion procedure (90 °C, overnight) was adopted for organic component and gold degradation. Iron concentration was estimated at 0.55 mg/L (~9.85 × 10⁻⁶ M). UV-vis analysis was performed on Cary Varian 50 Probe UV Spectrophotometer (Varian, Palo Alto, CA, USA). In all analyses, 1 cm path length quartz cuvettes were used. All the data were analyzed by using the Origin Pro 8 (Origin Lab Corporation, Northampton, MA, USA) and the Kaleidagraph software (version 4.1.1, Synergy Software, Reading, PA, USA).

All experiments were performed at least three times, the fits to data were generated by the Kaleidagraph software (Synergy). Fluorescence anisotropy-binding experiments were carried out in either 40 mM HEPES pH 7.4, 160 mM KCl, 5% glycerol, 0.1% Triton X-100, 1 mM EDTA, 5 mM DTT (herein referred to as ‘buffer A') or 40 mM HEPES pH 7.4, 400 mM KCl, 5% glycerol, 0.1% Triton X-100, 1 mM EDTA and 5 mM DTT (herein referred to as ‘buffer B') at room temperature as described in Morrone et al.23 (link)
Förster resonance energy transfer experiments were carried out in either buffer A (for AIM2^FL and AIM2^Hin) or buffer C (40 mM HEPES pH 7.4, 60 mM KCl, 5% glycerol, 0.1% Triton X-100, 1 mM EDTA and 5 mM DTT) for AIM2^Hin as described in Morrone et al.23 (link)
Electrophoretic mobility shift assay experiments were carried out in buffer A. To a fixed amount of fluorescein-labelled dsVACV72 was added increasing concentrations of AIM2. The reaction was allowed to equilibrate at room temperature (at least 20 min), then applied to a 4% 116:1 acrylamide:bis-acrylamide Tris-borate-EDTA gel. The gel was run at 100 V in 1 × Tris-borate-EDTA buffer and imaged using a Typhoon imager (GE Healthcare; excitation at 488 nm, emission at 532 nm).

Experimental results were obtained from at least three independent experiments. The differences were analyzed by a two-tailed Student’s t-test and labeled with asterisks as “statistically significant” if the probability, p, was less than 0.05 compared with control. GraphPad Prism software (GraphPad Software, La Jolla, CA, USA) was used for curve plotting, and KaleidaGraph software (Synergy Software, Reading, PA, USA) was used for statistical analysis.

An analysis of variance of the cell/animal response group was performed using the Kaleidagraph software (Synergy Software, Reading, PA, USA). Values are given as means ± SEM. Significant responses (P < 0.05) are marked by symbols (#, *) and their corresponding p-values are provided in figure legends. When non-specified experiments were performed in three to five times in triplicates.

Data were presented as mean ± standard error of the mean (SEM). All data were analyzed using Kaleida Graph software (version 4.5; Synergy software, Reading, PA, USA). The trapezoidal rule was used to determine the net incremental area under the curve (net AUC). The ‘t’ test was used to determine statistically significant differences. Statistical significance was set at P < 0.05.

Experimental data were determined from at least three independent experiments and the calculated IC₅₀ values were presented as mean ± standard deviation (SD) unless stated otherwise. The difference between the control value or experimental value or improvement in fit was analysed by two-tailed Student’s t-test. The difference was considered as “statistically significant” if the probability, p, was less than 0.05 and was labelled with asterisks. GraphPad Prism software (GraphPad Software, La Jolla, CA, USA) was used for curve plotting, whereas KaleidaGraph software (Synergy Software, Reading, PA, USA) was used for statistical analysis.

All data are presented as mean ± standard error using KaleidaGraph software (Synergy Software, Reading, PA, USA). Statistical significance was evaluated by a one-way ANOVA analysis for multiple comparison between groups. Statistical comparisons between two groups were conducted by a Student’s t-test. A p value of <0.05 was taken as significant.